| Background and ObjectiveAdenomyosis is a common benign disease of the uterus affecting 19.5%of women of reproductive age.Its histopathological features are ectopic endometrial tissue(endometrial glands and stroma)in the myometrium,surrounded by proliferative smooth muscle.Clinical manifestations include pelvic pain,abnormal uterine bleeding,and infertility.Despite its high incidence and severe symptoms,its pathogenesis and etiology remain unclear.There are two main theories that explain the origin and pathogenesis of adenomyosis.One theory relies on the concept of a dysfunctional tissue damage and repair mechanism that promotes cell migration and invagination of the basement of the endometrium into the muscular layer.Another theory suggests that adenomyosis is the result of metaplasia or differentiation of adult stem cells from displaced embryonic polypotent Mullerian remains.Although adenomyosis is a benign disease,it has the characteristics of invasion and metastasis of malignant tumors.Migration and invasion are thought to be key factors in the progression and spread of adenomyosis.Previous studies have shown that epithelial-mesenchymal transition(EMT)plays an important role in the invasion and metastasis of malignant tumors,and its association with the formation and progression of uterine adenomyosis has also been widely studied.EMT is a process in which epithelial cells change their phenotype,lose main epithelial cell characteristics,transform to express mesenchymal cell markers and gain invasion and metastasis ability.EMT can be observed in a variety of physiological and pathological environments,including normal embryogenesis and tissue morphogenesis,tissue repair and remodeling,fibrosis and tumor progression.A functional loss of expression of the cell adhesion marker E-cadherin and a concomitant increase in the expression of the interstitial markers N-cadherin and Vimentin in epithelial cells have been suggested as markers of EMT.In adenomyoma lesions,the expression of E-cadherin was decreased,and expression of Vimentin and N-cadherin was increased in epithelial cells compared with normal endometrium(control group),suggesting that the process supporting and promoting invasion and progression of adenomyoma lesions may be determined by EMT.However,the mechanisms that trigger EMT in adenomyosis have not been elucidated.Macrophages have different polarization tendencies in different microenvironments.Classical binary macrophage activation phenotypes include M1 and M2 types.M1 macrophages are classified as having a proinflammatory phenotype,while M2 is considered to be anti-inflammatory macrophages.Nitric oxide synthase 2(iNOS),TNF-α and IL-12 are usually used as Ml-type macrophage markers,while CD 163,Arginase 1(Arg-1),TNF-β and IL-10 are often considered as M2-type macrophage markers.Studies have shown that there are a large number of tumor-associated macrophages(TAM)in the tumor microenvironment,and their activation status is similar to M2 macrophages.TAM can produce a variety of cytokines,promote the survival,angiogenesis and metastasis of malignant tumor cells,and promote the occurrence of EMT.In adenomyosis,macrophages accumulated in the eutopic endometrium.Therefore,we infer that macrophages are involved in the formation of adenomyosis.miRNAs are single-stranded non-coding RNA that can bind to target mRNA and interfere with the translation process.It has been confirmed that there are a variety of miRNAs differentially expressed in endometriosis,and these miRNAs are involved in the development of endometriosis by targeting genes,yet the role of miRNAs in the pathogenesis of adenomyosis is still unclear.Extracellular vesicles(EVs)are small vesicles secreted by cells.They encase proteins,lipids,and genetic material,are present in biological fluids,allow cell-to-cell exchange,and are thought to be involved in cell-to-cell communication.Recent studies have demonstrated the involvement of extracellular vesicles(EVs)in tumor-associated macrophages(or M2-Polarized Macrophages)and EMT-associated events.miRNAs in EVs can regulate macrophage polarization and promote EMT,and act as signaling molecules that regulate tumor growth,angiogenesis,metastasis,chemotherapy sensitivity and immune evasion.In this study,we hypothesized that adenomyosis-derived EVs could promote macrophage polarization and promote EMT in endometrial epithelial cells of eutopic endometrium.Primary endometrial epithelial cells,Ishikawa cells,THP-1-derived macrophages and primary human peripheral blood-derived mononuclear macrophages were used as research objects in vitro to explore the possible role of adenomyosis-derived EVs and the miRNA that encapulated in EVs to the polarization of macrophage and pathogenesis of adenomyosis.We hope that this study would provide a new theoretical basis for the etiology and treatment of adenomyosis.Part Ⅰ Effect of adenomyosis-derived extracellular vesicles on the polarization state of macrophagesObjectiveOur aim was to extract extracellular vesicles from the culture supernatant of primary endometrial cells and investigate their effect on the polarization state of macrophages.Materials and Methods1.The accumulation of M2 macrophages in eutopic endometrium of patient with adenomyosis and women in control was verified by immunohistochemistryPremenopausal patients with adenomyosis who had not received hormone therapy for at least 3 months and had no systemic diseases and received hysterectomy or adenomyosis lesion resection plus diagnostic curettage were selected as the experimental group(n=21).Women with tubal obstructive infertility without any clinical indications or a history of adenomyosis or endometriosis,who underwent combined hysteroscopic and laparoscopic surgery or hysteroscopic surgery,were selected as the control group(n=10)(Qilu Hospital of Shandong University Scientific Research Ethics Committee,approval number:KYLL-2020(KS)-177).Eutopic endomembrane tissue was obtained from women in the experimental group and control group.After formalin fixation,the expression of CD 163 positive macrophages was detected by immunohistochemical staining.2.Isolation and identification of primary endometrial cell derived EVsThe primary endometrial stromal cells were isolated from patients with adenomyosis or women in control group with enzymatic hydrolysis method after collecting the eutopic endometrial tissue.Cells from passages 1～3 with vigorous viability were used in subsequent experiments.EVs were isolated from the culture supernatant of primary endometrial stromal cells by Exo-spinTM.EVs were identified by electron transmission microscopy,nanoparticle tracer analysis(NTA)and western blot.3.Culture and identification of macrophagesThe experimental subjects were macrophages derived from human THP-1 cell line and human primary mononuclear macrophages.THP-1 cells were stimulated by PMA and became macrophages,which were identified by immunofluorescence.Peripheral blood primary mononuclear cells(PBMC)were obtained by density gradient centrifugation after double dilution of human anticoagulant blood samples.Mononuclear cells were obtained by CD14+magnetic bead sorting method and cultured for 7 days in M-CSF to differentiate into primary monocyte-derived macrophages.Primary human peripheral blood mononuclear macrophages were identified by immunofluorescence assay.4.Verification of the uptake of EVs by THP-1 derived macrophages and primary mononuclear macrophagesThe EVs labeled with red PKH26 were treated with macrophages for 12 hours.Then macrophages were labeled with CD68,the nuclei were stained with DAPI dye,and the phagocytosis of macrophages was observed by fluorescence microscope.5.The effect of EVs on the polarization of macrophages was detected by qRT-PCR and Enzyme-Linked Immunosorbent Assay(ELISA)EVs from patients with adenomyosis,EVs from women in control group,and the culture supernatants of primary endometrial stromal cells with or without GW4869(non-competitive inhibitor of neutral sphingomyelinase),were treated on THP-1-derived macrophages and human primary monocyte-derived macrophages,respectively.After 48 hours,total RNA was extracted and the mRNA contents of CD163,IL-10,TNF-α and iNOS were detected.The concentration of cytokines in the supernatant of macrophages was detected by ELISA.Results1.M2 macrophages accumulate in the eutopic endometrium of adenomyosisThe results of immunohistochemistry showed that CD 163-positive macrophages were aggregated in the eutopic endometrium of adenomyosis patients compared with the control group.2.Identification of EVsTransmission electron microscopy(TEM)showed that EVs were round or oval,dark in the center and light in the outer ring.It looks like a saucer,with a brighter ring around it.NTA showed that most of the particles were between 50 and 200nm in diameter.Western blot showed that EVs highly expressed CD63 and TSG101,but not Calnexin.After adding the EVs inhibitor GW4869,the expression levels of CD63,TSG101 and CD81 proteins in EVs decreased.3.Identification of macrophagesTHP-1-derived macrophages and primary monocytic macrophages expressed the macrophage marker CD68,which was labeled with red fluorescence in THP-1-derived macrophages and green in primary monocytic macrophages.Nuclei are all marked in blue.After the two types of macrophages were attached to the wall,some cells grew tentacles,and the tentacles of primary monocytic macrophages were more obvious.4.Macrophages can take up EVsUnder the fluorescence microscope,the EVs marked as red by PKH26 gathered in the membrane(green)and around the nucleus(blue)of macrophages.5.Adenomyosis-derived EVs can induce macrophage polarization toward M2bTHP-1-derived macrophages highly expressed CD163,IL-10,and TNF-α mRNA after stimulation with adenomyosis-derived EVs,and iNOS mRNA showed a decreasing trend,but not statistically significant.After stimulation with the culture supernatant of primary endometrial stromal cells from adenomyosis,THP-1-derived macrophages showed high expression of CD163,IL-10 and TNF-α mRNA,and low expression of iNOS mRNA.After addition of GW4869,the mRNA of iNOS was highly expressed,but the expression of mRNA of CD163,IL-10 and TNF-α was not significantly increased.Primary human monocyte-derived macrophages showed the same trend of polarization as THP-1-derived macrophages.ELISA showed that the concentrations of IL-10 and TNF-α cytokines in the culture supernatant of THP-1-derived macrophages were increased after stimulation with adenomyosis-derived EVs.Conclusion1.CD 163-positive macrophages accumulated in the eutopic endometrium of patients with adenomyosis compared with control women.2.Adenomyosis-derived EVs can induce macrophage polarization toward M2b.Part Ⅱ Study on promotion of EMT of primary endometrial epithelial cells by M2b macrophagesObjectiveOur aim was to investigate the changes of EMT-related protein expression and migration ability of endometrial epithelial cells after co-culture with M2b macrophages.Materials and Methods1.Detection of the EMT markers of glandular epithelial cells in the eutopic endometrium by Immunohistochemical methodWe obtained the eutopic endometrial tissue from patients with adenomyosis and women of control group,then detected the expressions of Vimentin and E-cadherin by immunohistochemical staining method.2.Identification of primary endometrial epithelial cells by immunofluorescenceThe primary endometrial epithelial cells were obtained by enzyme digestion method from eutopic endometrial tissue of patients with adenomyosis or women in control group.Compared with primary endometrial stromal cells,epithelial cells are less numerous and cannot be passaged,so fresh tissue extraction is required.The primary endometrial epithelial cells expressed epithelial marker CK7 and were labeled with green by immunofluorescence method,and nuclei were marked in blue with DAPI.3.Macrophages stimulated by EVs were co-cultured with epithelial cells,and the expression of EMT markers in epithelial cells was detected by Western blotEVs-treated macrophages were co-cultured with primary endometrial epithelial cells of patients with adenomyosis or women in control group for 48 hours.Western blot was used to detect the protein expression of N-cadherin,Vimentin,E-cadherin and CK7 in primary endometrial epithelial cells after co-culture.EVs-treated macrophages were co-cultured with Ishikawa cells for 48 hours.The protein expression of N-cadherin and E-cadherin in Ishikawa cells was detected by Western blot.4.Macrophages stimulated by EVs were co-cultured with epithelial cells,and the changes in the migration ability of epithelial cells were detected by Transwell assayThe changes of migration ability of primary endometrial epithelial cells and Ishikawa cells were detected by Transwell method using 24-well plates and the matched TranswellTM chamber.5.Macrophages stimulated by EVs were co-cultured with epithelial cells,and the expression of PTEN,p-AKT and AKT in epithelial cells was detected by Western blotThe co-culture method was the same as step 3,and the expressions of PTEN,p-AKT and AKT in primary endometrial epithelial cells and Ishikawa cells were detected by Western blot.Results1.EMT exists in the eutopic endometrial glandular epithelial cells of patients with adenomyosisImmunohistochemical staining showed that the expression of Vimentin was up-regulated,and E-cadherin was down-regulated in the glandular epithelial cells of the eutopic endometrium of patients with adenomyosis compared with the control group.2.Identification of primary endometrial epithelial cellsUnder fluorescence microscopy,the cytoplasm of the epithelial cells was stained green,and the nucleus was stained blue.When the number of cells was small,the primary endometrial epithelial cells grew in clusters and form rose-shaped cell clusters.When the number of cells was large,the primary endometrial epithelial cells could cover the culture plate.3.Macrophages stimulated by adenomyosis-derived EVs induced EMT in primary endometrial epithelial cells and Ishikawa cellsCompared with the co-culture of macrophages treated with EVs from control group,the protein expression of N-cadherin and Vimentin was increased,while the expression of E-cadherin and CK7 was decreased in primary endometrial epithelial cells after co-culture with macrophages treated with EVs from adenomyosis.The protein expression of N-cadherin in Ishikawa cells increased,and E-cadherin decreased after co-culture with macrophages treated with EVs from adenomyosis.4.Macrophages stimulated by adenomyosis-derived EVs enhanced the migration ability of primary endometrial epithelial cells and Ishikawa cellsCompared with macrophages treated with EVs from control group,the migration ability of primary endometrial epithelial cells from patients with adenomyosis and women in control and Ishikawa cells was enhanced after co-culture with macrophages treated with EVs from adenomyosis.5.When epithelial cells underwent EMT,PTEN expression was down-regulated and p-AKT/AKT expression was up-regulatedWhen macrophages stimulated by adenomyosis-derived EVs were co-cultured with primary endometrial epithelial cells from patients with adenomyosis,or primary endometrial epithelial cells from control group,or Ishikawa cells,the expression of PTEN in epithelial cells was down-regulated,and the ratio of p-AKT/AKT was up-regulated.The results suggested that the occurrence of EMT was related to the downregulation of PTEN and the activation of AKT pathway.Conclusion1.EMT existed in the eutopic endometrial glandular epithelial cells of patients with adenomyosis.2.Macrophages treated with adenomyosis-derived EVs could induce EMT in primary endometrial epithelial cells of women in the experimental group and the control group and Ishikawa cells.3.EMT of epithelial cells was related to the downregulation of PTEN and activation of AKT pathway.Part Ⅲ MiR-25-3p in EVs from adenomyosis induced polarization of the macrophages,and the polarized macrophages promoted EMT of epithelial cellsPurposeOur aim was to screen and identify the differentially expressed miRNA from the candidate miRNA in EVs of adenomyosis and control group and to verify the role of the miRNA in polarization of macrophage and EMT of primary endometrial epithelial cells.Materials and Methods1.Isolation and identification of EVs from serumEleven patients were randomly selected from the experimental group of Part I and Part Ⅱ as the experimental group(n=11)of this part;the original control patients of Part Ⅰ and Part Ⅱ were still used as the control group of this part of the study(n=10),and serum samples were collected.Serum derived EVs were extracted according to the instruction of Exo-spinTM.The morphology of EVs was observed by transmission electron microscopy,the particle size of EVs was detected by NTA,and the expression of TSG101,CD63 and Calnexin in EVs was detected by Western blot.2.Detection of the expression level of miR-21,miR-25-3p and miR-301a-3p in macrophages stimulated by adenomyosis-derived EVs and control EVsAccording to the research conclusions of the first two parts and previous literature,miR-21,miR-25-3p and miR-301a-3p were selected as the initial research objects.QRT-PCR was used to detect the expression of miR-21,miR-25-3p,and miR-301a-3p in macrophages stimulated by adenomyosis-derived EVs and EVs from control group.3.Detection of the expression of miR-21 and miR-25-3p in EVs derived from serum of patients with adenomyosis and EVs derived from serum of control groupAccording to the results of the previous step,qRT-PCR was used to detect the expression of miR-21 and miR-25-3p in EVs derived from serum of patients with adenomyosis and EVs from serum of the control group.4.Detection of the expression of miR-25-3p in adenomyosis-derived EVs and EVs from control groupAccording to the results of the previous step,qRT-PCR was used to detect the expression of miR-25-3p in adenomyosis-derived EVs and control EVs.5.Detection of the transfection efficiency after miR-25-3p mimics or inhibitor were transfected into macrophagesAfter transfection of miR-25-3p mimics or inhibitor into macrophages,the increase or decrease of miR-25-3p expression in macrophages were detected by qRT-PCR.6.Detection of the expression of polarization markers in macrophage after transfection with miR-25-3p mimics or inhibitorMiR-25-3p mimics or inhibitor were transfected into macrophages,and the mRNA expressions of CD 163,IL-10,TNF-α and iNOS were detected by qRT-PCR;the concentrations of IL-10 and TNF-α in the culture supernatant were detected by ELISA.7.Detection of the expression of PTEN in macrophages transfected with miR-25-3p mimics or inhibitorAfter transfection of miR-25-3p mimics or inhibitor to macrophages,the expression of PTEN in transfected macrophages was detected by Western blot.8.Detection of the expression of EMT markers in epithelial cells after co-culture with macrophages transfected with miR-25-3p mimics or inhibitorAfter transfection of miR-25-3p mimics or inhibitor into macrophages,the transfected macrophages were co-cultured with primary endometrial epithelial cells from patients with adenomyosis or primary endometrial epithelial cells from control group,then Western blot was used to detect the protein expression of N-cadherin,Vimentin,E-cadherin and CK7 in primary endometrial epithelial cells.MiR-25-3p mimics or inhibitor were transfected into macrophages and then co-cultured with Ishikawa cells,and the expression of N-cadherin and E-cadherin in Ishikawa cells was detected by Western blot.9.Detection of the changes in the migration ability of epithelial cells after co-culture with macrophages transfected with miR-25-3p mimics or inhibitorAfter transfection of miR-25-3p mimics or inhibitor into macrophages,the transfected macrophages were co-cultured with primary endometrial epithelial cells from patients with adenomyosis,primary endometrial epithelial cells from control group,or Ishikawa cells,then Transwell assay was used to detect the changes of migration ability of primary endometrial epithelial cells and Ishikawa cells.10.Detection of the expression of PTEN,p-AKT and AKT in epithelial cells after co-culture with macrophages transfected with miR-25-3p mimics or inhibitorAfter transfection of miR-25-3p mimics or inhibitor into macrophages,the transfected macrophages were co-cultured with primary endometrial epithelial cells from patients with adenomyosis,primary endometrial epithelial cells from control group,or Ishikawa cells,then the expressions of PTEN,p-AKT and AKT in primary epithelial cells and Ishikawa cells were detected by Western blot.Results1.Identification of serum derived EVsTransmission electron microscopy showed that the appearance of serum derived EVs was similar to that of saucer,and there was no significant difference between the appearance of serum derived EVs and that of cell supernatant derived EVs.NTA showed that the average diameter of EVs was 143.2±50.2 nm and the average concentration was 2.7*10^10 particles/ml;and the concentration of EVs extracted from serum was higher than that of EVs extracted from cell supernatant.Western blot showed that EVs highly expressed CD63 and TSG101,but not Calnexin.2.Macrophages stimulated by EVs derived from adenomyosis highly expressed miR-25-3p and miR-21MiR-25-3p and miR-21 were highly expressed in macrophages treated with EVs derived from adenomyosis,and miR-301a-3p mRNA showed an upward trend without statistical significance.3.Serum derived EVs from patients with adenomyosis highly expressed miR-25-3pCompared with the control group,miR-25-3p was highly expressed in serum EVs of patients with adenomyosis.miR-21 tended to increase,but it was not statistically significant.4.The EVs derived from the supernatant of primary stromal cells in the eutopic endometrium of adenomyosis highly expressed miR-25-3pCompared with EVs derived from culture supernatant of primary endometrial stromal cells from control women,EVs derived from culture supernatant of primary endometrial stromal cells from patients with adenomyosis highly expressed miR-25-3p.5.Detection of efficiency of macrophages transfected with miR-25-3p mimics or inhibitorCompared with the control group of mimics,the expression of miR-25-3p in macrophages transfected with miR-25-3p mimics was significantly increased.Compared with the control group of the inhibitor,the expression of miR-25-3p in macrophages transfected with inhibitor was significantly suppressed.The results indicated that transfection was effective.6.MiR-25-3p induced polarization of macrophages towards M2After miR-25-3p mimics were transfected into macrophages,qRT-PCR showed that macrophages had high expression of CD 163,IL-10 and Arg-1 mRNA,and low expression of TNF-α and iNOS mRNA.ELISA showed increased expression of IL-10 and decreased expression of TNF-α.Macrophages were polarized towards M2.After miR-25-3p inhibitor was transfected into macrophages,qRT-PCR showed decreased expression of CD 163,IL-10 and Arg-1 mRNA,and increased expression of TNF-α and iNOS mRNA.ELISA showed decreased expression of IL-10 and increased expression of TNF-α.Macrophages were polarized towards M1.7.MiR-25-3p might target PTEN in macrophagesAfter transfection with miR-25-3p mimics,the expression of PTEN in macrophages was down-regulated.Conversely,the expression of PTEN in macrophages was up-regulated after transfection with miR-25-3p mimics.8.The polarized macrophages induced by miR-25-3p could promote EMT of epithelial cellsAfter macrophages were transfected with miR-25-3p mimics and co-cultured with primary endometrial epithelial cells or Ishikawa cells,the protein expressions of N-cadherin and Vimentin in primary endometrial epithelial cells were increased,while the protein expressions of E-cadherin and CK7 were decreased;the expression of N-cadherin was increased,and E-cadherin was down-regulated in Ishikawa cells.9.The polarized macrophages induced by miR-25-3p could enhance the migration ability of epithelial cellsAfter macrophages were transfected with miR-25-3p mimics and co-cultured with primary endometrial epithelial cells or Ishikawa cells,the migration ability of primary endometrial epithelial cells and Ishikawa cells was enhanced.10.PTEN and AKT pathways may participate in the EMT process of epithelial cellsWhen EMT occurs,the expression of PTEN in primary endometrial epithelial cells and Ishikawa cells was down-regulated or tends to be down-regulated,and the ratio of p-AKT/AKT was up-regulated or tends to be up-regulated.Conclusion1.The expression of miR-25-3p was upregulated in adenomyosis-derived EVs,and miR-25-3p can induce macrophage polarization to M2.2.Macrophages induced by miR-25-3p could in turn promoted EMT in endometrial epithelial cells.3.There might be a variety of differentially expressed miRNAs in EVs derived from the eutopic endometrium of adenomyosis and EVs from the eutopic endometrium of the control group. |
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